Award Date

August 2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Committee Member

Ronald Gary

Second Committee Member

Gary Kleiger

Third Committee Member

Paul Forster

Fourth Committee Member

Boo Shan Tseng

Number of Pages

320

Abstract

Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine kinase involved in several key signaling pathways, including glycogen metabolism, WNT/β-catenin, and Hedgehog signaling. Hyperactivity of GSK3β has been linked to Alzheimer’s disease, bipolar disorder, type II diabetes, and some cancers. Therefore, GSK3β is of interest as a target for therapeutics. Lithium ion (Li+) is a classical inhibitor of GSK3β. Structurally similar beryllium ion (Be2+) is ~1000-fold more potent. Lithium and beryllium have demonstrated pathway-specific and cell type-specific inhibition of GSK3β, prompting an investigation into the inhibitory mechanisms and thermodynamic characteristics of the metal-enzyme interaction.Isothermal titration calorimetry (ITC) is a label-free technique that measures the heat absorbed or released during binding interactions. Be2+ is hypothesized to inhibit GSK3β by competing with magnesium ions (Mg2+) for binding sites rich in acidic residues like aspartic acid. ITC binding studies utilizing model chelators and peptides featuring carboxylate groups revealed that Be2+ has a higher binding affinity than Mg2+ for carboxylate-rich binding sites. ITC can be extended to studying enzyme kinetics by measuring the heat released or absorbed during enzymatic reactions. This allows for real-time observation of reaction kinetics. This study reports the first use of an ITC enzyme assay for the kinetic analysis of a protein kinase. The ITC enzyme assay facilitates continuous monitoring of the enzyme reaction and provides mechanistic insights into the enzymatic process that may remain uncovered in conventional kinase assays. An ITC assay was developed using the single-injection method for the kinetic characterization of human GSK3β kinase activity. The assay was utilized to study the phosphorylation of a primed GSK3β substrate, the GSM phosphopeptide. The GSM phosphopeptide contains three tandem phosphorylation sites. Mutated forms of GSM, where specific phosphorylation sites were altered to alanine, were created to produce mono-site and zero-site GSM substrates. Phosphorylation of the mono-site GSM by GSK3β was an exothermic reaction and followed Michaelis-Menten kinetics, with a KM of 59 μM and kcat of 6.3 s-1. The intrinsic enthalpy of the reaction was -16 kJ/mol. The kinetic analysis for triple-site GSM phosphorylation did not conform to classical Michaelis-Menten kinetics and instead displayed a biphasic behavior. GSK3β sequentially phosphorylates substrates with multiple phosphorylation sites in tandem. A comparative analysis of reaction enthalpies and the shapes of the raw ITC thermograms for mono-site and triple-site GSM demonstrated that tandem multi-site phosphorylation by GSK3β follows a processive mechanism. The processive phosphorylation by GSK3β displays two modes: fast kinetics for ES complex formation and phosphorylation of the first site and slow kinetics for the phosphorylation of secondary and subsequent sites. This observation shows that once GSK3β initiates phosphorylation, it remains associated with the substrate to ensure complete modification of all tandem phosphorylation sites, following a mechanism designed to ensure reliability rather than speed. This provides insight into how the need for complete sequential phosphorylation by GSK3β plays a role in tightly controlled signaling pathways.

Keywords

GSK3β; ITC; ITC Kinase Assay; Processive Phosphorylation

Disciplines

Analytical Chemistry | Biochemistry | Biophysics

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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